High Throughput Screen to Identify Inhibitors of the Golgi GOLPH3 Pathway PROJECT SUMMARY GOLPH3 is the first example of an oncogene that functions in trafficking from the Golgi to the plasma membrane. The GOLPH3 gene is frequently amplified in human cancers (e.g. 56% of lung cancers) and overexpression of GOLPH3 drives oncogenic transformation in cell culture and xenograft models of cancer. Furthermore, overexpression of GOLPH3 occurs at high frequency in a wide spectrum of cancers and high expression predicts poor patient prognosis. We discovered GOLPH3 as a novel effector of the phosphoinositide PI4P. We showed that GOLPH3 localizes to the Golgi by binding to PI4P. GOLPH3 also binds to an unconventional myosin, MYO18A, which links the Golgi to the actin cytoskeleton. The PI4P/GOLPH3/MYO18A/F-actin pathway functions in Golgi-to-plasma membrane trafficking by applying a tensile force to assist in vesicle budding from the Golgi. This tensile force also helps to give the Golgi its familiar appearance by light and electron microscopy. Thus, the shape of the Golgi is a consequence of the mechanism of trafficking. The ability of GOLPH3 to drive oncogenic transformation in model systems is dependent on the PI4P/GOLPH3/MYO18A/F-actin pathway. Furthermore, we have shown that this GOLPH3 pathway can confer resistance to killing by DNA damaging agents, thus providing at least one explanation for the poor prognosis associated with high GOLPH3 expression. Taken together, the data predict that inhibitors of the GOLPH3 pathway will have efficacy against cancer and will synergize with conventional therapeutics. Our mechanistic studies have allowed us to develop highly sensitive and specific assays for GOLPH3 pathway activity. Our preliminary data validate our primary assay for a high throughput screen to identify small molecule inhibitors of the pathway and further demonstrate our ability to validate, stratify, and determine the molecular targets of hit compounds. Here we propose a high throughput screen to identify inhibitors of the Golgi GOLPH3 pathway to serve as tools to study the role of the Golgi GOLPH3 pathway in cancer and as lead compounds for novel cancer therapeutics.